Optimized Set Of Oligonucleotides For Bulk RNA Barcoding And Sequencing

Abstract

The present invention relates generally to the field of nucleic acid sequencing and provides oligonucleotide molecules and barcodes contained therein. These oligonucleotide molecules and barcodes molecules are useful in sequencing to identify and resolve errors.

1 . One or more oligonucleotide molecules comprising, from 5′ to 3′,
 a) a sequencing adapter, 
 b) a barcode sequence consisting of 9 to 15 nucleotides, 
 and, 
 c) an mRNA capture sequence. 
 
     
     
         2 . The one or more oligonucleotide molecules of  claim 1 , further comprising d) a unique molecular identifier (UMI). 
     
     
         3 . The one or more oligonucleotide molecules of  claim 1 , wherein the sequencing adapter comprises SEQ ID NO: 97. 
     
     
         4 . The one or more oligonucleotide molecules of  claim 1 , wherein the barcode sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 96 and a combination of one or more thereof. 
     
     
         5 . The one or more oligonucleotide molecules of  claim 2 , wherein the UMI consists of a sequence (N)n(V)m, wherein N is any nucleotide selected from A, T, C and G; V is any nucleotide selected from A, C and G; n is an integer selected from 1 to 20, and m is an integer selected from 1 to 20. 
     
     
         6 . The one or more oligonucleotide molecules of  claim 1 , wherein the mRNA capture sequence is a poly-T sequence followed by at least one V and one N, wherein N is any nucleotide selected from A, T, C and G and V is any nucleotide selected from A, C and G. 
     
     
         7 . A set of oligonucleotide molecules consisting of 96 oligonucleotide molecules, each molecule comprising, from 5′ to 3′,
 a) a sequencing adaptor, 
 b) a barcode sequence independently selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 96, 
 c) an mRNA capture sequence and, 
 d) optionally a UMI. 
 
     
     
         8 . A barcode oligonucleotide having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 96 and a combination of one of more thereof. 
     
     
         9 . (canceled) 
     
     
         10 . A method for making a cDNA library, the method comprising:
 a) Providing a plurality of RNA samples obtained from a biological sample;   b) Contacting separately each RNA sample with one or more oligonucleotide molecules of  claim 1  under annealing conditions;   c) Incubating separately each sample under reverse transcription reaction conditions;   d) Pooling together all the cDNA:RNA sample;   e) Proceeding to second strand synthesis under synthesis conditions; and   f) Proceeding with tagmentation and/or end-repair and ligation and amplification under suitable conditions so as to obtain a cDNA library.   
     
     
         11 . The method for making a cDNA library of  claim 10 , wherein the second strand synthesis is generated by a method comprising PCR amplification, nick translation, or a combination thereof. 
     
     
         12 . A method for sequencing RNA, the method comprising the steps of
 a) Providing a cDNA library obtained by the method of  claim 10 ; and   b) Proceeding to sequencing under suitable conditions.   
     
     
         13 . A method for selecting barcode oligonucleotides for multiplexed nucleic acid sequencing, the method comprising selecting one or more barcode oligonucleotides which has/have a Shannon's first order entropy of at least 1.5 and a second order entropy of at least 2.5; and wherein
 GC content of the barcode oligonucleotides is comprised between 35% to 65%;   the first two nucleotides are not G's;   the last two nucleotides are not T's;   the hamming distance between two barcodes of the list is at least 5; and   said one or more barcode oligonucleotides contain homopolymers of maximum 2 nucleotides.   
     
     
         14 . The method of  claim 13 , wherein the method is a computer implemented method. 
     
     
         15 . A kit comprising:
 i) one or more oligonucleotide molecules of  claim 1 ,   ii) a support for sample preparation, and   iii) reagents for sequencing.   
     
     
         16 . (canceled) 
     
     
         17 . The one or more oligonucleotide molecules of  claim 1 , wherein the barcode sequence consists of 12 nucleotides. 
     
     
         18 . The one or more oligonucleotide molecules of  claim 3 , wherein the sequencing adaptor consists of SEQ ID NO: 97.